基于无性期18S rDNA特异性引物检测5种疟原虫qPCR的建立和应用

doi:10.12140/j.issn.1000-7423.2023.01.006

摘要/Abstract

摘要：

目的建立基于疟原虫无性期（A期）18S rDNA检测感染人的5种疟原虫的qPCR，并评价其检测效果。方法从GenBank中下载5种疟原虫A期、子饱子期（S期）、卵囊期（O期）和人的18S rDNA序列进行比对，划分保守区和变异区，在保守区设计疟原虫属特异性引物，在变异区设计种特异性引物。以5种疟原虫DNA、田鼠巴贝虫和婴儿利什曼原虫DNA为模板进行qPCR扩增，筛选特异性引物对。应用qPCR法筛选特异性引物对的最佳退火延伸温度和引物浓度。分别构建5种原虫A期18S rDNA短片段（筛选出的引物对扩增产物）和长片段（属特异性引物扩增产物）质粒，以连续浓度梯度的短片段质粒DNA[（5 × 10¹）~（5 × 10⁸）拷贝/μl]、长片段质粒DNA[（5 × 10⁴）~（5 × 10⁹）拷贝/μl]为模板分别进行qPCR扩增，测定筛选出的引物对的扩增效率、最低检出限[包括最低质粒浓度、循环数阈值（Ct值）、变异系数]、无扩增时非目标DNA的最高浓度、可检出的混合DNA中不同虫种间的最大拷贝浓度比值。以5倍稀释的18份疟原虫血样DNA[恶性疟原虫（Pf）6份、间日疟原虫（Pv）6份、卵形疟原虫（Po）4份、三日疟原虫（Pm）2份、诺氏疟原虫（Pk）1份]为模板，应用筛选出的特异性引物分别进行qPCR、一步反转录qPCR和巢式PCR扩增，计算最低检出限时的原虫密度。用筛选出的特异性引物进行应用验证，对70份疟疾患者血样DNA（Pf 26份、Pv 14份、Pm 14份、Po 9份、Pk 3份、混合感染4份）、65份疟原虫阴性人血样DNA以及阿米巴（1份）、田鼠巴贝虫（1份）和杜氏利什曼原虫DNA（3份）进行qPCR扩增，以样品提供者的检测结果为标准，计算qPCR检测的敏感度、特异度、符合率。不同PCR方法检测结果的比较采用卡方检验、t检验、Mann-Whitney检验或Wilcoxon秩和检验。结果序列比对结果显示，5种疟原虫A期18S rDNA序列的一致性为89.3%~96.7%，高于S期的60.0%~92.1%，可分为9个保守区和8个变异区。除Pv O期和Pm S期18S rDNA外，保守区序列长度相同，一致性≥ 92%。筛选获得1对属特异性引物和5对种特异性引物，各引物对的qPCR扩增效率为90.6%~98.8%，qPCR扩增的最佳退火延伸温度为57.8℃，最佳引物终浓度为0.3 μmol/L。6对特异性引物的qPCR检出的最低质粒浓度均为5 × 10²拷贝/μl，对应Ct值为32.3~33.1，变异系数为0.5%~3.7%。5对种特异性引物qPCR扩增非目标DNA结果均为阴性时非目标DNA的最高浓度为（5 × 10⁶）~（5 × 10⁸）拷贝/μl，可检出的混合质粒中不同虫种间的最大拷贝浓度比值均为10⁴。一步反转录qPCR、qPCR和巢式PCR可检出的最低原虫密度平均值分别为（4.1 ± 5.1）、（6.0 ± 4.3）和（8.2 ± 2.9）虫/μl血，最低值分别为0.01、0.3和2.0虫/μl血，变异系数分别为125.6%、72.2%和35.8%，一步反转录qPCR检出最低原虫密度的敏感性高于另两种方法（Wilcoxon秩和检验，Z = -2.0、-2.0，均P < 0.05），qPCR和巢式PCR的敏感性差异无统计学意义（t = -1.7，P > 0.05）。应用验证结果显示，qPCR的敏感度和特异度均为98.6%，qPCR检测结果与标准结果的符合率为98.6%，差异无统计学意义（χ² = 0.0，P > 0.05）。结论建立了基于疟原虫A期18S rDNA的qPCR检测系统，可用于鉴定常规疟疾血样。

关键词: 疟原虫, 染料法qPCR, 一步反转录qPCR, 最低检出限

Abstract:

Objective To establish a qPCR method for detecting the 18S rDNA of the asexual stage (stage A) of 5 Plasmodium species and to evaluate its detection efficacy. Methods The 18S rDNA sequences of stage A, sporozoite stage （stage S） and oocyst (stage O) of five human malaria parasite species and the sequence from human were downloaded from GenBank and aligned to confirm the distribution pattern of the conserved and variable regions. Specific primers of Plasmodium genus were designed in the conserved regions and the specific primers of species were designed in the variable regions. Specific primer pairs were screened by the qPCR amplifying the DNA of the five Plasmodium species, Microtus babesi and Leishmania infantis as templates, and the optimal annealing extension temperature and primer concentration of the specific primer pairs were screened. The plasmids of short 18S rDNA fragments (amplified with screened primers) and long 18S rDNA fragments (amplified DNA fragments with conserved primers) of the five Plasmodium species of stage A were constructed respectively. The qPCR amplification was carried out with plasmid DNA [(5 × 10¹） - (5 × 10⁸) copy/μl] of the short fragments and plasmid DNA [(5 × 10⁴）- (5 × 10⁹) copy/μl] of the long fragment as templates, and the amplification efficiency, the minimum detection limit [including the minimum plasmid concentration and the corresponding cycle threshold (Ct) value, the coefficient of variation of repeated detection], the maximum concentration of non-target DNA when there was no amplification, and the maximum copy concentration ratio between the different Plasmodium species in the detectable mixed DNA were determined respectively. Using the DNA of the 18 blood samples infected with Plasmodium parasites (6 samples of P. falciparum, 6 samples of P. vivax, 2 samples of P. malariae, 4 samples of P. ovale, and 1 sample of P. knowlesi), which were diluted by 5 folds as the template, the selected specific primers were used for qPCR and one-step reverse transcription qPCR amplification respectively, and nested PCR amplification was performed at the same time to calculate and compare the density of the samples at the lowest detection limit. Using selected specific primers, qPCR amplification was performed on the DNA from 70 malaria patients’ blood samples (26 samples of P. falciparum, 14 samples of P. vivax, 14 samples of P. malariae, 9 samples of P. ovale, 3 samples of P. knowlesi, 4 samples of mixed infections), 65 whole blood samples of malaria-negative patients, and Amoeba sp. (1 sample), M. babesi (1 sample), and Leishmania donovani (3 samples) samples to calculate the sensitivity, specificity and conformity. Chi-square test, t-test, Mann-Whitney test or Wilcoxon rank sum test were used to compare the results of different PCR methods. Results Sequence alignment results showed that the consistency of the 18S rDNA sequence of the five Plasmodium species of parasites in stage A was 89.3%-96.7%, higher than those in stage S (60.0%-92.1%). Nine conserved regions and eight variable regions were mapped. Except for the 18S rDNA fragments of stage O of P. vivax and stage S of P. malraiae, the sequence length of the conserved regions was the same with consistency of ≥ 92%. One pair of generic primers and 5 pairs of specific species primers were selected for detecting the five species of human malaria parasites. When amplified with qPCR, the amplification efficiency of all primer pairs were 90.6%-98.8%, the optimal annealing extension temperature was 57.8 ℃, and the optimal final primer concentration was 0.3 μmol/L. The minimum detection limit for the short fragment plasmid was 5 × 10² copies/μl, the corresponding Ct value is 32.3-33.1 and the coefficient of variation of repeated detection is 0.5%-3.7%. The highest concentration of non-target DNA was (5 × 10⁶)-(5 × 10⁸) copies/μl when no amplification presented. The maximum copy concentration ratio between different Plamodium species in the mixed DNA that can be detected reached 10⁴. The average minimum paparsite density detected by one-step reverse transcription qPCR, qPCR and nested PCR were (4.1 ± 5.1), (6.0 ± 4.3) and (8.2 ± 2.9) parasites/μl respectively, and the lowest detected protozoa density was 0.01, 0.3 and 2.0 parasites/μl respectively, and the coefficient of variation were 125.6%, 72.2% and 35.8%, respectively. The sensitivity of the one-step reverse transcription qPCR is higher than those of the other two methods (Wilcoxon rank sum test, Z = -2.0, -2.0; P < 0.05). There was no statistically significant difference in the sensitivity between qPCR and nested PCR (t = -1.7, P > 0.05). The results of practical application showed that, the sensitivity and specificity of the qPCR were both 98.6%. The coincidence rate between the application results of the qPCR and the standard results was 98.6%, and their difference was not statistically significant (χ² = 0.0, P > 0.05). Conclusion The qPCR detection system using specific primers based on the 18S rDNA of Plasmodium spp. asexual stage has been established which can be applied in identifying malaria parasites in blood samples.

Key words: Plasomodium, Dye-qPCR, One-step qPCR, Detection threshold

中图分类号:

R382.31

李美, 肖宁, 夏志贵. 基于无性期18S rDNA特异性引物检测5种疟原虫qPCR的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 36-43.

LI Mei, XIAO Ning, XIA Zhigui. Establishment and application of a specific primers-based qPCR targeting asexual stage 18S rDNA for detecting five Plasmodium species[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(1): 36-43.

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保守区编号 Code of conservative area	在Pf-A上的位置 Location on Pf-A	片段长度/bp Fragment length/bp	一致性/% Identification/%
保守区编号 Code of conservative area	在Pf-A上的位置 Location on Pf-A	片段长度/bp Fragment length/bp	Po-S	Pf-S	其他10条 Other 10 sequences
C1	1~65	65	95	98	100
C2	85~126	42	98	98	98
C3	298~480	183	96	98	≥ 99
C4	550~644	95	99	98	100
C5	942~1 100	159	93	94	≥ 97
C6	1 184~1 454	271	98	99	100
C7	1 638~1 724	87	92	97	100
C8	1 831~1 978	148	92	97	≥ 98
C9	2 003~2 092	90	92	97	100

引物名称 Primer name		引物序列（5´→3´） Primer sequence (5´→3´)	在18S rDNA上的位置 Location on 18S rDNA	片段长度/bp Fragment length/bp	Tm值^a/℃ Tm value^a /℃	扩增效率/% Amplification efficiency/%
疟原虫属 Plasmodium spp.	Pg-F	GCGCAAGCGAGAAAGTTAAAAG	C6	145	82	98.8
疟原虫属 Plasmodium spp.	Pg-R	CCGCTAATTAGCAGGTTAAGAT	C6	145	82	98.8
Pf	Pf-F	TTATTATCCTTTGATTTTTATCTTTGG	V2	108	72	90.6
Pf	Pf-R	ATAAATTTATTACGTGTTACTTCTTTG	V2	108	72	90.6
Pv^[17]	Pv-F	TTTTAAGGACTTTCTTTGCTTCGG	V3	321	79.5 ~ 80.0	92.7
Pv^[17]	Pv-R	CGACGGTATCTGATCGTCTTC	C4	321	79.5 ~ 80.0	92.7
Pm	Pm-F	CAAGCGTTATATTTTTTCTGTTACAT	V3	165	73.5 ~ 74.5	96.0
Pm	Pm-R	GAAACTGTTTTAACTGTTTGCTTTG	V3	165	73.5 ~ 74.5	96.0
Po	Po-F	TATAAGATGCTTAGGCAATACAAC	V3	177	76.0 ~ 77.5	97.3
Po	Po-R	TCAAACGCAGTAAAATGCTTTAACT	V3	177	76.0 ~ 77.5	97.3
Pk	Pk-F	GAAGAAAATATTGGAATTACGTTAAAT	V5	90	73.5 ~ 74.0	95.7
Pk	Pk-R	CATTCATACGCAAAAAAGAAATAGAT	V5	90	73.5 ~ 74.0	95.7

引物对 Primer pairs	非目标DNA的最高浓度/拷贝·μl^-1The highest concentration of non-target DNA/copy·μl^-1
引物对 Primer pairs	Pf	Pv	Pm	Po	Pk
Pf-F/Pf-R		5.0 × 10⁸	5.0 × 10⁷	5.0 × 10⁷	5.0 × 10⁷
Pv-F/Pv-R	5.0 × 10⁸		5.0 × 10⁸	5.0 × 10⁸	5.0 × 10⁷
Pm-F/Pm-R	5.0 × 10⁶	5.0 × 10⁶		5.0 × 10⁶	5.0 × 10⁷
Po-F/Po-R	5.0 × 10⁷	5.0 × 10⁶	5.0 × 10⁶		5.0 × 10⁷
Pk-F/Pk-R	5.0 × 10⁶	5.0 × 10⁶	5.0 × 10⁷	5.0 × 10⁷

引物对 Primer pairs	目标DNA Target DNA				目标和非目标DNA混合 Mix of target DNA and non-target DNA
	5.0 × 10²拷贝·μl^-1 5.0 × 10²copy·μl^-1		5.0 × 10⁶拷贝·μl^-1 5.0 × 10⁶copy·μl^-1		5.0 × 10²拷贝·μl^-1 5.0 × 10²copy·μl^-1
	平均Ct值 Average Ct values	变异系数/% Coefficient of variation/%	平均Ct值 Average Ct values	变异系数/% Coefficient of variation/%	非目标DNA浓度/拷贝·μl^-1 Concentration of non-target DNA/copy·μl^-1	平均Ct值 Average Ct values	变异系数/% Coefficient of variation/%
Pf-F/Pf-R	34.1 ± 0.8	2.5	19.9 ± 0.0	0.1	5.0 × 10⁶	34.3 ± 0.1	0.3
Pv-F/Pv-R	30.6 ± 0.5	1.5	17.5 ± 0.0	0.0	5.0 × 10⁷	31.6 ± 0.9	3.0
Pm-F/Pm-R	32.1 ± 0.0	0.1	18.4 ± 0.1	0.4	5.0 × 10⁶	32.6 ± 0.4	1.4
Po-F/Po-R	31.7 ± 0.7	2.1	17.6 ± 0.0	0.0	5.0 × 10⁷	31.4 ± 0.4	1.2
Pk-F/Pk-R	33.6 ± 0.1	0.3	19.4 ± 0.0	0.0	5.0 × 10⁶	33.4 ± 0.4	1.2

引物对 Primer pairs	样品 Sample	最低检出原虫密度/虫·μl^-1 Minimum parasite density/Parasite·μl^-1
引物对 Primer pairs	样品 Sample	一步反转录qPCR One-step reverse transcription qPCR	qPCR	巢式PCR Nest PCR
Pg-F/Pg-R	X差值^a最大的恶性疟原虫样品 P. falciparum sample with maximum D-value of X^a	0.08	2.00
Pf-F/Pf-R	X差值最大的恶性疟原虫样品 P. falciparum sample with maximum D-value of X	0.02	2.00	9.9
Pf-F/Pf-R	X差值最小的恶性疟原虫样品 P. falciparum sample with mimimum D-value of X	0.40	0.40	2.0
Pv-F/Pv-R	X差值最大的间日疟原虫样品 P. vivax sample with maximum D-value of X	0.01	0.30	6.9
Pv-F/Pv-R	X差值最小的间日疟原虫样品 P. vivax sample with mimimum D-value of X	10.80	10.80	10.8
Pm-F/Pm-R	X差值最大的三日疟原虫样品 P. malariae sample with maximum D-value of X	0.30	7.40	7.4
Pm-F/Pm-R	X差值最小的三日疟原虫样品 P. malariae sample with mimimum D-value of X	10.6	10.60	10.6
Po-F/Po-R	X差值最大的卵形疟原虫样品 P. ovale sample with maximum D-value of X	0.06	7.70	7.7
Po-F/Po-R	X差值最小的卵形疟原虫样品 P. ovale sample with mimimum D-value of X	10.00	10.00	10.0
Pk-F/Pk-R	诺氏疟原虫样品 P. knowlesi sample	8.30	8.30
	平均最低检出限 Everage detection limit	4.1 ± 5.1	6.0 ± 4.3	8.2 ± 2.9